This project has two long-term goals: First, to characterize the biochemical properties of the LexA protein of Escherichia coli, and second, to identify and characterize other cellular proteins which interact with activated RecA protein. The LexA protein is a repressor of a set of genes, often termed the LexA regulon, which is controlled by the SOS regulatory system. This system is activated when cellular DNA is damaged. The key event in controlling the SOS response is a specific proteolysis of LexA protein. Cleavage inactivates the repressor. In vitro, LexA protein also has an intrinsic protease activity ("autodigestion") which cleaves the molecule in an intramolecular reaction. Autodigestion and RecA-dependent cleavage appear to be closely related. The work is aimed at characterizing these cleavage reactions and the relationsip between them, using a combination of genetic and biochemical techniques. Mutants will be isolated and sequenced which impair or enhance the reaction. Mutant proteins will be isolated and their ability to take part in the cleavage reactions will be determined. Pseudorevertants of these mutants will be characterized. In addition, the structure of LexA protein will be studied, using recombinant DNA techniques to alter its structure. Its binding to its specific operators will be further characterized, methods will be developed to assess accurately the relative affinities for different binding sites, and co-operative binding to adjacent operators will be studied. Interaction of LexA protein with activated RecA protein will be characterized. Efforts will be made to identify and characterize other cellular proteins which also interact with activated RecA protein, an important goal because the mutagenesis process induced by the SOS system required activated RecA protein. These experiments thus will test the model that activated RecA plays its role in mutagenesis by binding to other proteins, perhaps those involved in DNA replication, and modifying their function. This work is important for two diverse reasons: First, characterizing LexA protein should give insights into protein structure and function, as well as specific DNA binding. Second, mutagenesis is important in carcinogenesis and a biochemical understanding of this process in bacteria offers the best approach to the problem, since we can integrate genetic and biochemical approaches.